MYLOTARG® (gemtuzumab ozogamicin) consists of a monoclonal antibody against CD33 that is bound to calicheamicin by means of an acid-hydrolyzable linker. The commercial product was marketed as the first antibody-targeted chemotherapeutic agent and was approved for the treatment of acute myeloid leukemia (AML) in elderly patients. Inotuzumab ozogamicin is a CD22 antibody linked to a calicheamicin currently in clinical trials for treatment of certain types of cancer.
The potent family of antibacterial and antitumor agents known collectively as the calicheamicins, or the LL-E33288 complex, are disclosed in U.S. Pat. Nos. 4,970,198 and 5,053,394 also discloses methyltrisulfide antibacterial and antitumor agents. These compounds in U.S. Pat. Nos. 4,970,198 and 5,053,394 contain a methyltrisulfide group that can be reacted with appropriate thiols to form disulfides while at the same time introducing a functional group such as a hydrazide or similar nucleophile. Examples of this reaction with the calicheamicins are given in U.S. Pat. Nos. 5,053,394 and 5,770,70 is directed to a process for preparing targeted forms of disulfide compounds of the LL-E33288 complex. A linker, 4-(4-acetyl-phenoxy)butanoic acid, is condensed with an N-acetyl gamma calicheamicin dimethyl hydrazide compound to afford the carboxylic acid-hydrazone which is further treated with N-hydroxysuccinimide to give the OSu ester (N-succinimidyloxy) which is ready for conjugation with a chosen biomacromolecule.
U.S. Pat. No. 8,273,862 describes a synthetic method for constructing linker intermediate molecules (termed “trilinker-activated esters” or “trifunctional linker intermediates” therein). These linker intermediates can be conjugated to calicheamicins to prepare calicheamicin derivatives that can then be further conjugated to biomacromolecules such as monoclonal antibodies. In the steps leading to the preparation of the linker intermediate, the synthetic method described in U.S. Pat. No. 8,273,862 uses a mercapto compound (“compound 2” therein), for example 3-methyl-3-mercaptobutanoic acid hydrazide, as an intermediate.
WO 2008/147765 describes a method for synthesizing the mercapto-containing intermediates, such as 3-methyl-3-mercaptobutanoic acid hydrazide, that are useful in preparing linker intermediates and calicheamicin derivatives as described in the preceding paragraph. WO 2008/147765 alternatively refers to the 3-methyl-3-mercaptobutanoic acid hydrazide as the “DMH linker”. WO 2008/147765 notes that 3-methyl-3-mercaptobutanoic acid hydrazide is a preferred mercapto-containing N-acylhydrazine for the purpose of linking calicheamicin to monoclonal antibodies to make, for instance, gemtuzumab ozogamicin or inotuzumab ozogamicin.
In WO 2008/147765, 3-methyl-3-mercaptobutanoic acid hydrazide is prepared by removing the benzyl protecting group from the compound p-methoxybenzylthioether hydrazide under acidic conditions. In order to obtain p-methoxybenzylthioether hydrazide, WO 2008/137765 teaches first reacting p-methoxybenzylthioether acid with oxalyl chloride in methylene chloride to form p-methoxybenzylthioether acid chloride. The p-methoxybenzylthioether acid chloride is then added to a mixture of anhydrous hydrazine and methylene chloride to obtain the p-methoxybenzylthioether hydrazide. However, as is described in WO 2008/147765, the two reactants p-methyloxybenzylthioether acid and p-methoxybenzylthioether acid chloride themselves together generate an undesired by-product, bis-methoxybenzylthioether hydrazide, resulting in lower yield and quality. Furthermore, as described in WO 2008/147765, due to the reactive and unstable nature of the acid chloride molecule p-methoxybenzylthioether acid chloride, anhydrous hydrazine and low temperatures, e.g. −70° C., must be employed.
Another aspect in the preparation of calicheamicin derivatives involves chemically connecting the calicheamicin to the linker molecule. In order to conjugate the calicheamicin to the linker intermediate in U.S. Pat. No. 8,273,862, a final reaction between a calicheamicin and a “trilinker activated ester” (or “trifunctional linker intermediate”) is conducted. Structurally, calicheamicins contain a trisulfide moiety, as explained above, that is used in their derivatization, and the chemistry of this reaction between the trisulfide moiety of the calicheamicin and the trilinker activated ester is important in realizing good yields and purity. Previously used sulfur exchange reactions for calicheamicin derivatives have given complex reaction mixtures, multiple byproducts, and low yields.
Finally, the calicheamicin derivative is to be purified after its formation. The process of U.S. Pat. No. 8,273,862 involves purification of the calicheamicin derivative comprising a normal phase chromatography step. The normal phase chromatography step in the purification process described in U.S. Pat. No. 8,273,862 uses methylene chloride as a solvent, and, as stated above, exposure to methylene chloride above various minimum levels is considered to pose potential health hazards. When using normal phase chromatography including methylene chloride, precautions to limit exposure to methylene chloride must therefore be taken.